The following definitions are provided to facilitate an understanding of the present invention.
The term "Vibrio" as used herein, refers to the bacteria classified as such in Bergey's Manual of Systematic Bacteriology (N. R. Krieg [ed.], 1984, 516-538, Williams & Wilkins). Detection of Vibrio is important in various medical and public health contexts. The Vibrio are important agents of human disease. Vibrio bacteria can cause a variety of pathological conditions ranging from simple gastroenteritis to more severe illnesses.
The term "target" or "target molecule," in a diagnostic sense, refers to a molecule of interest, i.e. the molecule whose presence one wishes to Know. In a therapeutic sense, the term "target" or "target molecule" refers to a molecule associated with a disease or with an organism causing a disease.
The term "biological binding pair" as used in the present application refers to any pair of molecules which exhibit mutual affinity or binding capacity. A biological binding pair is capable of forming a complex under binding conditions. For the purposes of the present application, the term "ligand" will refer to one molecule of the biological binding pair, and the term "antiligand" or "receptor" will refer to the opposite molecule of the biological binding pair. For example, without limitation, embodiments of the present invention have application in nucleic acid hybridization assays where the biological binding pair includes two complementary nucleic acids. One of the nucleic acids is designated the ligand and the other nucleic acid is designated the antiligand or receptor. One of the nucleic acids may also be a target molecule. The designation of ligand or antiligand is a matter of arbitrary convenience. The biological binding pair may include antigens and antibodies, drugs and drug receptor sites, and enzymes and enzyme substrates, to name a few.
The term "probe" refers to a ligand of known qualities capable of selectively binding to a target antiligand or receptor. As applied to nucleic acids, the term "probe" refers to nucleic acid having a base sequence complementary to a target nucleic acid. The probe and the target are capable of forming a probe target complex under binding conditions. The term "probe" will be used herein, in both a diagnostic sense, meaning capable of binding a molecule, the presence or absence of which one desires to know, and a therapeutic sense, capable of binding to a molecule associated with a disease.
The term "label" refers to a chemical moiety which is capable of detection including, by way of example, without limitation, radioactive isotopes, enzymes, luminescent agents, precipitating agents, and dyes. The term "agent" is used in a broad sense, including any chemical moiety which participates in reactions which lead to a detectable response. The term "cofactor" is used broadly to include any chemical moiety which participates in reactions with the label.
The term "amplify" is used in the broad sense to mean creating an amplification product, which may include by way of example, additional target molecules, or target-like molecules, capable of functioning in a manner like the target molecule, or a molecule subject to detection steps in place of the target molecule, which molecules are created by virtue of the presence of the target molecule in the sample. In the situation where the target is a polynucleotide, additional target, or target-like molecules, or molecules subject to detection can be made enzymatically with DNA or RNA polymerase.
The term "contiguous" means an adjacent area of a molecule. By way of example, in the case of biological binding pairs, where a first ligand binds to a receptor target molecule, the area surrounding and adjacent to the first ligand is open and capable of binding to a second ligand contiguous to the first. In the context of nucleic acid, where a first probe binds to an area of a larger nucleic acid target molecule, an adjacent mutually exclusive area along the length of the target molecule can bind to a second probe which will then be contiguous to the first. The target molecule acts as a template, directing the position of the first probe and the second probe. The term "substantially contiguous" is used in the functional sense to include spatial orientations which may not touch, may not abut, or may overlap, yet function to bring parts, areas, segments and the like into cooperating relationship.
The term "capture ligand" means a ligand capable of specifically binding with a capture antiligand associated with a support.
The term "support" when used alone, includes conventional supports such as filters, dipsticks and membranes as well as retrievable supports.
Genetic information is stored in living cells in thread-like molecules of DNA. In vivo, the DNA molecule is a double helix of two complementary strands of DNA, each strand of which is a chain of nucleotides. Each nucleotide is characterized by one of four bases: adenine (A), guanine (G), thymine (T), and cytosine (C). The bases are complementary in the sense that, due to the orientation of functional groups, certain base pairs attract and bond to each other through hydrogen bonding and .pi.-stacking interactions. Adenine in one strand of DNA pairs with thymine in an opposing complementary strand. Guanine in one strand of DNA pairs with cytosine in an opposing complementary strand. In RNA, the thymine base is replaced by uracil (U) which pairs with adenine in an opposing complementary strand. The genetic code of a living organism is carried upon the DNA strand, in the sequence of base pairs.
Molecules of DNA consist of covalently linked chains of deoxyribonucleotides and molecules of RNA consists of covalently linked chains of ribonucleotides. Each nucleic acid is linked by a phosphodiester bridge between the. 5'-hydroxyl group of the sugar of one nucleotide and the 3'-hydroxyl group of the sugar of an adjacent nucleotide. The terminal ends of nucleic acid are often referred to as being 5'-termini or 3'-termini in reference to the terminal functional group. Complementary strands of DNA and RNA form antiparallel complexes in which the 3'-terminal end of one strand is oriented and bound to the 5'-terminal end of the opposing strand.
Nucleic acid hybridization assays are based on the characteristic of two nucleic acid strands to pair at their complementary regions to form hybrids. The formation of such hybrids can be made to be highly specific by adjustment of the conditions (sometimes referred to as stringency) under which this hybridization takes place such that hybridization will not occur unless the sequences are precisely complementary. If total nucleic acid from the sample is immobilized on a solid support such as a nitrocellulose membrane, the presence of a specific "target" sequence in the sample can be determined by the binding of a complementary nucleic acid "probe" which bears a label. After removal of non-hybridized probe by washing the support, the amount of target is determined by the amount of detectable moiety present.
Nucleic acid probes by design or selection, contain specific nucleotide sequences that allow them to hybridize under hybridization conditions, specifically and preferentially, to target nucleic acid sequences. The term "preferentially" is used in a relative sense, one hybridization reaction product is more stable than another under identical conditions. Under some conditions, a hybridization reaction product may be formed with respect to one target, but not to another potential binding partner.
Although, nucleic acid compositions of great length, up to 2500 nucleotides, have been suggested for use as probes, there are practical considerations which would suggest a smaller nucleic acid would have some advantage. A minimum of ten nucleotides are necessary in order to statistically obtain specificity and form stable hybridization products. A maximum of 250 nucleotides represents an approximate upper limit of sequences in which reaction parameters can be readily adjusted and controlled presently to determine mismatched sequences and preferential hybridization. The maximum 250 nucleotides also represent the upper limit of most DNA and RNA synthesis equipment. Most preferably, probe sequences have between 20 to 60 nucleotides.
Hybridization conditions are defined by the base composition of the probe/target duplex, as well as by the level and geometry of mispairing between the two nucleic acids. Normal hybridization conditions for nucleic acid of 10 to 250 bases are a temperature of approximately 60.degree. C. in the presence of 1.08M sodium chloride, 60 mM sodium phosphate, and 6 mM ethylenediamine tetraacetic acid (pH of 7.4).
Reaction parameters which are commonly adjusted include concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and the temperature of hybridization. Generally, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a corollary, the stringency of the conditions under which hybridization is to take place (e.g., based on the type of assay to be performed) will dictate certain characteristics of the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art.
Ribosomes are of profound importance to all organisms. Ribosomes serve as the only known means of translating genetic information into cellular proteins, the math structural and catalytic elements of life. A clear manifestation of this importance is the observation that all cells have ribosomes.
Bacterial ribosomes contain three distinct RNA molecules which, at least in Escherichia coli, re referred to as 5S, 16S and 23S rRNAs. In eukaryotic organisms, there re four distinct rRNA species, generally referred to as 5S, 18S, 28S, and 5.8S. These names historically are related to the size of the RNA molecules, as determined by their sedimentation rate. In actuality, however, ribosomal RNA molecules vary substantially in size between organisms. Nonetheless, 5S, 16S, and 23S rRNA generic names for the homologous RNA molecules in any bacterium and this convention will be continued herein. Detailed discussion of the 16S and 23S rRNA primary and secondary structures my be found in Gutell, ei.al. (Progress in Nucleic Acid Research, vol. 32, 1985) and Gutell and Fox (Nucleic Acids Research, vol. 16 supplement, 1988).
Kohne et al. (1968) Biophysical Journal 18:1104-1118 discuss one method for preparing probes to rRNA sequences.
Pace and Campbell, Journal of Bacteriology 107:543-547 (1971), discuss the homology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantitating such homology levels. Similarly, Sogin et al., Journal of Molecular Evolution 1:173-184 (1972), discuss the theoretical and practical aspects of using primary structural characterization of different ribosomal RNA molecules for evaluating phylogenetic relationships.
Fox et al., International Journal of Systematic Bacteriology (1977), discuss the comparative cataloging of 16S ribosomal RNAs as an approach to prokaryotic systematics.